The nuclear hormone receptor (NHR) family of transcription factors bind low molecular weight ligands and either stimulate or repress transcription. (in The Nuclear Receptor Facts Book, V. Laudet and H. Gronemeyer, Academic Press, p 345, 2002). NHRs stimulate transcription by binding to DNA and inducing transcription of specific genes. NHRs may also stimulate transcription by not binding to DNA itself, rather they may modulate the activity of other DNA binding proteins (Stocklin, E., et al., Nature (1996) 383:726-8). The process of stimulation of transcription is called transactivation. NHRs repress transcription by interacting with other transcription factors or coactivators and inhibiting the ability of these other transcription factors or coactivators to induce transcription of specific genes. The process of repression of transcription is called transrepression. (for a review see The Nuclear Receptor Factsbook, V. Laudet and H. Gronemeyer, Academic Press, p 42, 2002). For example, the glucocorticoid receptor, estrogen receptor, androgen receptor and peroxisome proliferator activated receptors α and γ have been shown to repress the activity of the transcription factors AP-1 and NF-κB (Jonat, C., et al., Cell, 62, p 1189-1204, (1990) Kallio, P. J., et al., Mol. Endocrinol., 9, p 017-1028 (1995), Keller, E. T., et al., J. Biol. Chem., 271, p 26267-26275 (1996), Jones, D. C., et al., J. Biol. Chem., 277 (9), p 6838-6845, (2002), Ricote, M., et al., Nature, 391, p 79-82, (1998), Valentine, J. E., et al, J. Biol. Chem., 275, p 25322-25329, (2000).
The nuclear hormone receptor family includes the glucocorticoid receptor (Hollenberg, S. M. et al. (1985) Nature, 318, p 635), progesterone receptor (Misrahi, M. et al. (1987) Biochem. Biophys. Res. Commun. 143, p 740), androgen receptor (Lubahn D. B., et al (1988), estrogen receptors (Green, S., et al. (1986) Nature 320, p 134), mineralocorticoid receptor (Arriza, J. L., et al., (1987) Science 237, p 268), retinoid receptors (RXRs and RARs) (Mangelsdorf, et al. (1990) Nature, 345, p 224 and Petkovich M., et al (1987) Nature 330, p 444), Vitamin D receptor, thyroid receptor (TR) (Nakai, A. et al., (1988) Mol. Endocrinol. 2, p 1087), peroxisome proliferator activated receptor (PPAR) (Greene, M. E., et al. (1995) Gene Expression 4, p 281), orphan nuclear receptors and others. Glucocorticoid receptor, progesterone receptor, androgen receptor, estrogen receptor, and mineralocorticoid receptor are steroid hormone receptors (SHRs).
Although the sequences vary amongst the various nuclear hormone receptors, they can be divided into functional domains including an N-terminal transactivation domain, a central DNA binding domain and a C-terminal ligand and dimerization domain. The ligands which bind these receptors act in a ligand, cell type, and promoter dependent fashion and include: glucocorticoids, progestins, retinoids, mineralocorticoids, and others. In addition to steroids, recent studies have shown that non-steroids can bind to nuclear hormone receptors and induce a biologic response (Coghlan, M J, et al, J. Med. Chem. 44, p 2879, 2001). Ligand cross-talk can occur between the receptors, for example, progesterone can bind not only the progesterone receptor but the glucocorticoid receptor as well (Zhang, S., Mol. Endocrinology 10, p 24, 1996).
Three-dimensional structures of some of the nuclear hormone receptors have been elucidated through crystallization or homology modeling. A homology model of the glucocorticoid receptor is disclosed in WO 00/52050, published Sep. 8, 2000.
Recent publications by the same research group: Bledsoe, et. al., Cell, online publication by Cell Press, Jul. 1, 2002, DOI: 10.1016/S0092867402008176; Cell, Vol 110, 93-105, 12 Jul. 2002; and Apolito, et. al., in WO 03/015692 A2, published Feb. 27, 2003; describe the successful crystallization and xray structural elucidation of the glucocorticoid receptor LBD as the dimer. X-ray structure coordinates were provided in WO 03/015692. Disruption of the dimeric structure was found to occur upon mutation of selected residues at the dimerization interface. Despite structural similarity to other steroid receptors, the GR LBD dimer represents a unique dimer configuration. The GR LBD used for this crystalization was a mutant (F602S) designed to provide a more soluble LBD construct.
Also recently, Kauppi et. al. published the stucture of the GR LBD bound to an antagonist, RU-486, in: the Journal of Biological Chemistry Online, JBC Papers In Press as DOI:10.1074/JBC.M212711200, Apr. 9, 2003; and in J. Biol. Chem., Vol. 278, Issue 25, 22748-22754, Jun. 20, 2003. In this structure, the GR LBD exhibits a significant displacement of helix 12, typical of antagonist action. In addition to the antagonist-bound LBD, a dimer structure similar to that reported by Bledsoe, et. al. was also described. The structure of the GR LBD-RU-486 complex was deposited in with the RCSB (1nhz.pdb)
Three dimensional structures of other nuclear hormone receptors are disclosed as follows, with RCSB (Research Collaboratory for Structural Bioinformatics, pdb file format) references in parentheses: RXRalpha (1lbd) Bourguet, W., Ruff, M., Chambon, P., Gronemeyer, H., Moras, D. Nature 375 pp. 377 (1995); PPAR-gamma (2prg) Nolte, R. T., Wisely, G. B., Westin, S., Cobb, J. E., Lambert, M. H., Kurokawa, R., Rosenfeld, M. G., Willson, T. M., Glass, C. K., Milburn, M. V. Nature 395 pp. 137 (1998); RARgamma (2lbd) Renaud, J. P., Rochel, N., Ruff, M., Vivat, V., Chambon, P., Gronemeyer, H., Moras, D. Nature 378 pp. 681 (1995); PR (1a28) Williams, S. P., Sigler, P. B. Nature 393 pp. 392 (1998); VitDR (1db1) Rochel, N., Wurtz, J. M., Mitschler, A., Klaholz, B., Moras, D. Mol. Cell 5 pp. 173 (2000); AR (1e3g) Matias, P. M., Donner, P., Coelho, R., Thomaz, M., Peixoto, C., Macedo, S., Otto, N., Joschko, S., Scholz, P., Wegg, A., Basler, S., Schafer, M., Egner, U., Carrondo, M. A. J. Biol. Chem. 275 pp. 26164 (2000); ERalpha (1a52) Tanenbaum, D. M., Wang, Y., Williams, S. P., Sigler, P. B. Proc Natl Acad Sci USA 95 pp. 5998 (1998); ERbeta (1l2j) Shiau, A. K., Barstad, D., Radek, J. T., Meyers, M. J., Nettles, K. W., Katzenellenbogen, B. S., Katzenellenbogen, J. A., Agard, D. A., Greene, G. L. Nat. Struct. Biol. 9 pp. 359 (2002). It is generally thought that all steroid ligands bind to nuclear hormone receptors at the classical ligand binding site, which we term site I (Evans, R. M. Science 240, p 889, 1988). Limited proteolysis studies and cell transfection/mutagenesis studies have delineated the functional domains of nuclear hormone receptors which include a DNA binding domain, ligand binding domain and a transactivation domain. These studies provided the evidence that hormones bind to the ligand binding domain. Mutagenesis of GR has defined the dexamethasone interacting surface, defined as Site I, which includes amino acids Met-560, Met-639, Gln-642 and Thr-739 (Lind, U., et al. J. Biol. Chem. 275, p 19041, 2000).
Recently, a second ligand binding site in ER-α and ER-β has been reported based on computational analysis and docking experiments with steroids. (van Horn, W. J. Med. Chem. 45, p 584, 2002). This second binding site is not completely delineated. It is reported to have no obvious function, to be an evolutionary remnant, and to be absent in other nuclear receptors such as RARγ. Furthermore, there is no discussion of transrepression whatsoever. In addition, Endocrine Society Meeting June 2003, presentation OR34-1, Wang, Y., Chirgadze, N Y, Briggs, S L, Khan, S., Jensen, E V., Burris, T P., A second binding site for hydroxytamoxifen with the ligand binding domain of estrogen receptor beta, describes the crystal structure of estrogen receptor bound with 4-hydroxytamoxifen, in which the ligand is found in two locations: the usual steroid binding pocket and a second site located along the hydrophobic groove near the cofactor binding region. This second site is remote from the Site II location described in this application.
The glucocorticoid receptor (GR) is a member of the nuclear hormone receptor family of transcription factors, and a member of the steroid hormone family of transcription factors. Affinity labeling of the glucocorticoid receptor protein allowed the production of antibodies against the receptor which facilitated cloning the human (Weinberger, et al. Science 228, p 740-742, 1985, Weinberger, et al. Nature, 318, P635-641, 1985) and rat (Miesfeld, R. Nature, 312, p 779-781, 1985) glucocorticoid receptors. Subsequently, glucocorticoid receptors from other species were cloned including mouse (Danielson, M. et al. EMBO J., 5, 2513), sheep (Yang, K., et al. J. Mol. Endocrinol. 8, p 173-180, 1992), and marmoset (Brandon, D. D., et al, J. Mol. Endocrinol. 7, p 89-96, 1991). There is also a C-terminally distinct isoform of GR termed GR-beta. This isoform is identical to GR up to amino acid 727 and then diverges in the last C-terminal 15 amino acids. GR-beta is not known to bind glucocorticoids, is unable to transactivate, but does bind DNA (Hollenberg, S M. et al. Nature, 318, p 635, 1985, Bamberger, C. M. et al. J. Clin Invest. 95, p 2435, 1995). It is possible that GR-beta binds compounds other than the typical glucocorticoids.
Glucocorticoids which interact with GR have been used for over 50 years to treat inflammatory diseases. It has been clearly shown that glucocorticoids exert their anti-inflammatory activity via the inhibition by GR of the transcription factors NF-κB and AP-1. This inhibition is termed transrepression. It has been shown that the primary mechanism for inhibition of these transcription factors by GR is via a direct physical interaction. This interaction alters the transcription factor complex and inhibits the ability of NF-κB and AP-1 to stimulate transcription (Jonat, C., et al. Cell, 62, p 1189, 1990, Yang-Yen, H. F., et al. Cell 62, p 1205, 1990, Diamond, M. I. et al. Science 249, p 1266, 1990, Caldenhoven, E. et al., Mol. Endocrinol. 9, p 401, 1995). Other mechanisms such as sequestration of co-activators by GR have also been proposed (Kamer Y, et al., Cell 85, p 403, 1996, Chakravarti, D. et al., Nature 383, p 99, 1996). NF-κB and AP-1 play key roles in the initiation and perpetuation of inflammatory and immunological disorders (Baldwin, A S, Journal of Clin. Investigation 107, p 3, 2001, Firestein, G. S., and Manning, A. M. Arthritis and Rheumatism, 42, p 609, 1999, Peltz, G., Curr. Opin, in Biotech. 8, p 467, 1997). NF-κB and AP-1 are involved in regulating the expression of a number of important inflammatory and immunomodulatory genes including: TNF-alpha, IL-1, IL-2, IL-5, adhesion molecules (such as E-selectin), chemokines (such as Eoxtaxin and Rantes), Cox-2, and others.
Although glucocorticoids are very effective anti-inflammatory agents, their systemic use is limited by their side effects which include diabetes, osteoporosis, glaucoma, Cushingoid syndrome, muscle loss, facial swelling, personality changes, and others. (Stanbury, R M, and Graham, E M, Br. J. Opthalmology 82, p 704, 1998, Da Silva, J A P., Bijlsma, J. Rheumatic Disease Clinics of North America, 26, p 859, 2000)
In addition to leading to transrepression, the interaction of a glucocorticoid with GR can lead to stimulation by GR of transcription of certain genes. This stimulation of transcription is termed transactivation. Transactivation requires dimerization of GR and binding to a glucocorticoid response element (GRE). DNA binding is mediated via Zn fingers in the DNA binding domain (Giguere, V. et al Cell 46, p 645, 1986; Rusconi, S. and Yamamoto, K. R. EMBO J., 6, p 1309, 1987). DNA sequence specific interactions are determined by the C-terminal part of the first Zn finger (Danielsen, M., et al. Cell 57, p 1131, 1989). Several GR target genes have been identified including MMTV, metallothionein, and tyrosine amino transferase (Ringold, G M et al, Cell 6, p 299,1975; Scheidereit, C., et al Nature, 304, p 749, 1983; Hager, L J, Palmiter R D, Nature 291, 340, 1981; Grange, T., et al. Oncogene, 20, p 3028, 2001). Transrepression, as opposed to transactivation, can occur in the absence of dimerization, and as mentioned above is believed to involve the direct interaction of GR with AP-1 and NF-κB.
Recent studies using a transgenic GR dimerization defective mouse which cannot bind DNA have shown that the transactivation (DNA binding) activities of GR could be separated from the transrepressive (non-DNA binding) effect of GR. These studies also indicate that many of the side effects of glucocorticoid therapy are due to the ability of GR to induce transcription of various genes involved in metabolism, whereas, transrepression, which does not require DNA binding leads to suppression of inflammation. (Tuckermann, J. et al. Cell 93, p 531, 1998; Reichardt, H M. EMBO J., 20, p 7168, 2001).
Compounds which can induce transrepression of GR with none to minimal induction of transactivation have been termed “dissociated steroids” (Vayassiere, B M, et al., Mol. Endocrinology, 11, p 1249, 1997). Such “dissociated” compounds would be useful to treat inflammatory diseases. See FIG. 1 for a graphical description of transactivation mediated by GR dimers versus transrepression mediated by GR monomers. It is possible that these “dissociated” compounds bind to GR without inducing dimerization yet allow the monomer to transrepress AP-1 and NF-κB. Another plausible explanation is that “dissociated” compounds may alter the conformation of GR to enable transrepression without inducing a DNA binding conformation.
There are several examples in the literature of compounds that possess dissociated activity as defined by the ratio of the effective concentration required to induce DNA binding in a cellular assay relative to the effective concentration required to transrepress, or inhibit AP-1 or NF-κB activity. The first report of a “dissociated steroid” published by Vayssiere, et al. Molecular Endocrinology, 11, p 1245, 1997, showed that a derivative of dexamethasone had potent in vitro and in vivo anti-inflammatory activity with minimal induction of DNA binding. Subsequent studies (Coghlan, M J, et al., J. Med. Chem. 44, p 4481, 2001) have shown that non-steroidal compounds can bind to GR and elicit transrepressive activity with moderate transactivation activity. It is believed that each of the compounds described above act via the dexamethasone binding site.
Ursodeoxycholic acid (UDCA) has recently been shown to repress NF-κB activity via a GR mediated pathway. The compound appears to be “dissociated” as it does not induce DNA binding in a cellular assay. This compound, although acting in a GR dependent fashion, does not compete for dexamethasone binding to GR. Although direct binding of UDCA to GR has not been demonstrated, mutagenesis studies suggest that the ligand binding domain (LBD) of GR is required for activity. (Miura, T., J. Biol. Chem. 276, p 47371, 2001). However, these studies did not delineate the specific amino acids which are involved in UDCA activity.
The art is in need of modulators of NHRs. A modulator of an NHR may be useful in treating NHR-associated diseases, that is diseases associated with the expression products of genes whose transcription is stimulated or repressed by NHRs. For instance, the art is in need of modulators of NHR that induce inhibition of AP-1 and NF-κB, as such modulators would be useful in the treatment of inflammatory and immune associated diseases and disorders, such as osteoarthritis, rheumatoid arthritis, multiple sclerosis, asthma, inflammatory bowel disease, transplant rejection, and graft vs. host disease.
The art is in need of compounds that possess dissociated activity, as such compounds would be useful in treating inflammatory and immune associated diseases and disorders without exhibiting unwanted side effects. For instance, in the case of GR, although glucocorticoids are potent anti-inflammatory agents, their systemic use is limited by side effects. A dissociated compound that retained the anti-inflammatory efficacy of glucocorticoids while minimizing the side effects such as diabetes, osteoporosis and glaucoma would be of great benefit to a very large number of patients with inflammatory diseases.
The art is in need of compounds that antagonize transactivation. For instance, in the case of GR, such compounds may be useful in treating metabolic diseases associated with increased levels of glucocorticoid, such as diabetes, osteoporosis and glaucoma.
The art is in need of compounds that induce transactivation. For instance, in the case of GR, such compounds may be useful in treating metabolic diseases associated with a deficiency in glucocorticoid. Such diseases include Addison's disease.
In order to design compounds that modulate an NHR in specific ways, one needs to understand how ligands bind to an NHR and modulate the activity of the NHR.